As databases for polymorphic markers and disease causing mutations continue to grow, there is an increasing need for procedures that can screen nucleic acid sequences for the presence of known polymorphisms and mutations. Optimally, the procedure should be capable of analyzing multiple DNA sites simultaneously (including nucleic acid loci that are physically separated by great distances) for the presence of mutations or polymorphisms.
Current methods for determining the genetic constitution of individuals (genotyping) include oligonucleotide ligation, allele-specific oligonucleotide hybridization, and PCR-restriction fragment length analysis. All these methods require time consuming multiple manual steps. One alternative method of genotyping uses the melting temperature of fluorescent hybridization probes that hybridize to a PCR amplified targeted region of genome/nucleic acid sequence to identify mutations and polymorphisms.
The polymerase chain reaction (PCR) is a technique of synthesizing large quantities of a preselected DNA segment. The technique is fundamental to molecular biology and is the first practical molecular technique for the clinical laboratory. PCR is achieved by separating the DNA into its two complementary strands, binding a primer to each single strand, at the end of the given DNA segment where synthesis will start, and adding a DNA polymerase to synthesize the complementary strand on each single strand having a primer bound thereto. The process is repeated until a sufficient number of copies of the selected DNA segment have been synthesized. During a typical PCR reaction, double stranded DNA is separated into its single strands by raising the temperature of the DNA containing sample to a denaturing temperature where the two DNA strands separate (i.e. the "melting temperature of the DNA") and then the sample is cooled to a lower temperature that allows the specific primers to attach (anneal), and replication to occur (extend). In preferred embodiments a thermostable polymerase is utilized in the polymerase chain reaction. A preferred thermostable DNA polymerase for use in the PCR reaction is the Taq DNA Polymerase and derivatives thereof, including the Stoffel fragment of Taq DNA polymerase and KIenTaqI polymerase (a 5'-exonuclease deficient variant of Taq polymerase--see U.S. Pat. No. 5,436,149).
Thermocycling may be carried out using standard techniques known to those skilled in the art, including the use of rapid cycling PCR. Rapid cycling techniques are made possible by the use of high surface area-to-volume sample containers such as capillary tubes. The use of high surface area-to-volume sample containers allows for a rapid temperature response and temperature homogeneity throughout the biological sample. Improved temperature homogeneity also increases the precision of any analytical technique used to monitor PCR during amplification.
In accordance with the present invention amplification of a nucleic acid sequence is conducted by thermal cycling the nucleic acid sequence in the presence of a thermostable DNA polymerase. The method comprises the steps of placing a biological sample comprising the nucleic acid sequence in a capillary vessel, raising the temperature of the biological sample from a first temperature to a second temperature wherein the second temperature is at least 15.degree. C. higher than the first temperature, holding the biological sample at the second temperature for a predetermined amount of time, lowering the temperature of the biological sample from the second temperature to at least the first temperature and holding the biological sample at a temperature at least as low as the first temperature for a pre-determined length of time. The temperature of the biological sample is then raised back to the second temperature, and the biological sample is thermocycled a predetermined number of times. In one embodiment, the method of amplifying a DNA sequence comprises a two temperature cycle wherein the samples are cycled through a denaturation temperature and an annealing temperature for a predetermined number of repetitions. However the PCR reaction can also be conducted using a three temperature cycle wherein the samples are cycled through a denaturation temperature, an annealing temperature and an elongation temperature for a predetermined number of repetitions.
In one embodiment each temperature cycle of the PCR reaction is completed in approximately 60 seconds or less. Rapid cycling times can be achieved using the device and techniques described in U.S. Pat. No. 5,455,175, the disclosure of which is expressly incorporated herein.
In accordance with the present invention PCR amplification of one or more targeted regions of a DNA sample is conducted in the presence of a fluorescently labeled hybridization probes, wherein the probes are synthesized to hybridize to a specific locus present in a target amplified region of the DNA. In a preferred embodiment the hybridization probe comprises two oligonucleotide probes that hybridize to adjacent regions of a DNA sequence wherein each oligonucleotide probe is labeled with a respective member of a fluorescent energy transfer pair. In this embodiment the presence of the target nucleic acid sequence in a biological sample is detected by measuring fluorescent energy transfer between the two labeled oligonucleotides.
Fluorescence resonance energy transfer (FRET) occurs between two fluorophores when they are in physical proximity to one another and the emission spectrum of one fluorophore overlaps the excitation spectrum of the other. The rate of resonance energy transfer is EQU (8.785E.sup.-5) (t.sup.-1) (k.sup.2) (n.sup.-4) (q.sub.D) (R.sup.-6) (J.sub.DA),
where:
t=excited state lifetime of the donor in the absence of the acceptor; PA1 k.sup.2 =an orientation factor between the donor and acceptor; PA1 n=refractive index of the visible light in the intervening medium; PA1 q.sub.D =quantum efficiency of the donor in the absence of the acceptor; PA1 R=distance between the donor and acceptor measured in Angstroms; PA1 J.sub.DA =the integral of (F.sub.D) (e.sub.A) (W.sup.4) with respect to W at all overlapping wavelengths with:
F.sub.D =peak normalized fluorescence spectrum of the donor; PA2 e.sub.A =molar absorption coefficient of the acceptor (M.sup.-1 cm.sup.-1); PA2 W.sup.4 =wavelength (nm).
For any given donor and acceptor, a distance where 50% resonance energy transfer occurs can be calculated and is abbreviated R.sub.0. Because the rate of resonance energy transfer depends on the 6th power of the distance between donor and acceptor, resonance energy transfer changes rapidly as R varies from R.sub.0. At 2 R.sub.0, very little resonance energy transfer occurs, and at 0.5 R.sub.0, the efficiency of transfer is nearly complete, unless other forms of de-excitation predominate.
Fluorescence resonance energy transfer can be used as a labeling system for detecting specific sequences of DNA. In combination with standard melting curve analysis, single point mutations in a gene can be distinguished from the normal gene. In accordance with one embodiment such a detection system comprises two oligonucleotides that hybridize to adjacent loci on DNA. The oligonucleotides are each labeled, respectively, with one of the fluorophores of a fluorescent resonance energy transfer pair, so that upon hybridization of the two labeled oligonucleotides to their complementary sequences on the targeted DNA, resonant energy is transferred from the donor fluorophore to the acceptor fluorophore. Such an energy transfer event is detectable and is indicative of the presence of the target nucleic acid sequence.
The fluorescently labeled oligonucleotides are designed to hybridize to the same strand of a DNA sequence resulting in the donor and acceptor fluorophores being separated by a distance ranging from about 0 to about 25 nucleotides, more preferably about 0-5 nucleotides, and most preferably about 0-2 nucleotides. A particularly preferred spacing between the donor and acceptor fluorophores is about 1 nucleotide.
When one of the labeled oligonucleotides also functions as a PCR primer ("probe-primer"), then the two fluorescently labeled oligonucleotides hybridize to opposite strands of a DNA sequence. In this embodiment, the donor and acceptor fluorophores are preferably within about 0-15 nucleotides and more preferably within about 4-6 nucleotides.
When both of the fluorescently labeled oligonucleotides are not hybridized to their complementary sequence on the targeted DNA, then the distance between the donor fluorophore and the acceptor fluorophore is too great for resonance energy transfer to occur. Thus the acceptor fluorophore and the donor fluorophore are not in resonance energy transfer relationship and excitation of the donor fluorophore will not produce a detectable increased fluorescence by the acceptor fluorophore.
Acceptable fluorophore pairs for use as fluorescent resonance energy transfer pairs are well know to those skilled in the art and include, but are not limited to, fluorescein/rhodamine, phycoerythrin/Cy7, fluorescein/Cy5 or fluorescein/Cy5.5.
The thermal stability of a DNA duplex relies on duplex length, GC content, and Watson-Crick base pairing. Changes from Watson-Crick pairing destabilize a duplex by varying degrees depending on the length of the mismatched duplex, the particular mismatch, the position of the mismatch, and neighboring base pairs. Accordingly, the percent identity of the hybridization probes to their target complementary sequence directly impacts the temperature at which the hybridization probe will separate (melt) from the complementary strand. The greater the difference between the probe and the target complementary sequence the lower the temperature needed to separate the hybridizing strands. Accordingly, an oligonucleotide probe identical in sequence to the complementary wild type sequence will dissociate from the locus containing a mutation at a lower temperature than it will from the wild type locus. The use of fluorescently labeled hybridization probes enables dynamic monitoring of fluorescence as the temperature of the sample is raised and the melting curve for the hybridization probe is determined. The generated melting curve is then compared to the known melting curve for the normal, mutant or polymorphic sequence to determine the sequence of the target nucleic acid locus.